Modified PCT-HD Workflow: Simplified Protocol with Fewer Steps and Less Hands-on Time
The PCT-HD workflow (also known as PCT-SWATHTm or PCT-ABLE) is a combination of two of PBI’s innovative sample preparation tools; the 2320EXT Barocycler and PCT MicroPestles [1-4]. Together they utilize pressure cycling technology (PCT) to extract and digest proteins from biopsy-size tissue samples. PCT has been proven to accelerate enzymatic protein digestion; the positive effect of PCT on digestion by both trypsin and Lys-C has previously been demonstrated [2-9]. High hydrostatic pressure improves protein digestion via a combination of two mechanisms; one acting on the enzyme and the other acting on the substrates, leading to significantly accelerated and highly reproducible digestion (Figure 1). The method can be adjusted for digestion with trypsin alone, lys-C alone, or a combination of both enzymes. Additional optimization may be required when using non-conventional proteolytic enzymes. PCT-HD can be carried out using conventional enzymes or boutique enzymes designed for higher temperature methods .